The long term objective of this proposal is to understand, at the molecular level, the mechanisms by which human papillomavirus type 18 transforms epithelial cells. HPV-16 and 18 are the presumed etiological agents of cervical intraepithelial neoplasia. We have recently utilized an in vitro system (referred to as the "raft" system) for epithelial differentiation, to examine the effects of human papillomaviruses on keratinocytes. When the entire HPV-16 genome was transfected into either an immortalized human epithelial cell line, SCC-13, or into primary human foreskin cells, it was observed to inhibit normal morphological differentiation when examined in the raft system. The morphological changes induced by HPV-16 in vitro resemble those seen in genital cancers in vivo. During the next period we will concentrate on the mechanism of papillomavirus induced inhibition of differentiation. We will identify the viral gene products responsible for immortalization and inhibition of differentiation and then try to characterize their mechanism of action. We will: I. Establish that HPV-18 inhibits epithelial cell differentiation in a manner similar to HPV-16. II. Identify HPV-18 gene products responsible for immortalization and inhibition of differentiation. III. Identify important regions of HPV-18 transforming genes by deletion analysis and site directed mutagenesis. IV. Examine the effects of HPV-6 and 11 in this system. V. Examine the effects of cellular oncogenes such as ras and myc in cooperation with HPV-18. VI. Study the mechanism by which HPV-18 inhibits epithelial cell differentiation.